Despite extensive diabetes related research, the sequence of events which occur subsequent to the coupling of insulin to its cell surface receptor are not known. The objective of this project is to test the hypothesis that insulin receptor tyrosine kinase activity or autophosphorylation are involved in mediating the antilipolytic effect of insulin in adipocytes. The experimental approach will use digitonin-permeabilized adipocytes which, despite loss of freely diffusable cytosolic contents, have an intact lipolytic pathway which is inhibitable by physiological concentrations of insulin. An additional characteristic of this preparation which is of vital importance is the accessibility of the cytosolic compartment to experimental manipulation. With the long-term goal of characterizing the mechanism of insulin's antilipolytic effect, this proposal has four specific aims: 1) further characterize the insulin receptor phosphorylation reaction which has been demonstrated in permeabilized cells, and investigate coupling between receptors, their phosphorylation and antilipolysis, 2) examine regulation of both receptor phosphorylation and dephosphorylation by insulin and lipolytic regulators; analyze reactions through phosphoamino acid patterns and peptide mapping, and correlated observations with insulin's antilipolytic activity, 3) further characterize the reassembly of partially purified insulin receptors into pronase-treated, permeabilized adipocytes which has been demonstrated to restore antilipolysis; explore the elements of the receptor that are essential for insulin-dependent antilipolysis and 4) characterize insulin-dependent protein phosphorylation in permeabilized adipocytes and correlate with receptor autophosphorylation. Using data from both antilipolysis and protein phosphorylation, assess the potential role the receptor kinase might play in insulin action. This proposal is directed toward an understanding of initial signal transduction following insulin binding to adipocytes. Advancements in this area will increase our understanding of antilipolysis which is the most important response in adipose tissue to insulin. Additionally, by explaining the mechanism by which insulin mediates, this process, the knowledge may be applicable to other processes and target tissues affected by insulin.